PROJECT SUMMARY Protein post-translational modifications by ubiquitin and ubiquitin-like proteins represent vital mechanisms regulating protein quality and function that are integral to cardiomyocyte function and homeostasis. The overall goal of this proposal is to determine the function and underlying mechanism of a novel ubiquitin-like protein, Ubiquitin-fold modifier 1 (Ufm1), in the heart. Ufmylation covalently conjugates Ufm1 to target substrates via a Ufm1-specific E1 (Uba5)-E2 (Ufc1)-E3 (Ufl1) cascade. Through regulating the function of cellular proteins, ufmylation controls multiple cellular processes and physiological events, and have been implicated in a number of human diseases. Our pilot studies have for the first time identified a critical role for ufmylation in constraining pathological cardiac remodeling and provided novel mechanistic linkages between ufmylation and endoplasmic reticulum (ER) stress response. Ufmylation is dysregulated in cardiomyopathic hearts. Inhibition of ufmylation via targeted ablation of the E3 Ufm1 ligase 1 (Ufl1) in the heart caused cardiomyopathy during ageing and promoted propensity to heart failure in response to hemodynamic stress. ER stress coincided with the progression of cardiomyopathy in these mice, and pharmacological attenuation of ER stress ameliorated cardiac dysfunction following pressure overload in Ufl1-deficient hearts. Furthermore, Ufl1 controls the expression of Ufm1 binding protein 1 (Ufbp1), an ER-resident Ufm1 target. Depletion of Ufbp1 diminished Xbp-1 splicing, blunted Xbp-1s signaling and aggravated ER stress-induced cell injury, recapitulating most aspects of Ufl1 depletion. Moreover, ER stress promotes the binding of Ufbp1 to IRE1?, a key ER stress transducer that activates cardioprotective Xbp-1s signaling. These data collectively suggest that Ufbp1 acts downstream of Ufl1 to protect CMs against pathogenic insults and is a crucial regulator of IRE1a/Xbp-1s signaling in cardiomyocytes. Therefore, this proposal is to test the hypothesis that ufmylation protects against pathological cardiac remodeling by targeting Ufbp1 to activate the adaptive ER stress response in cardiomyocytes. To test this hypothesis, Aim 1 will define the pathophysiological roles of Ufbp1 in the heart; Aim 2 will identify molecular bases of how ufmylation activates the adaptive ER stress response signaling in cardiomyocytes; Aim 3 will elucidate the functional importance of Ufbp1 ufmylation in activating IRE1a/Xbp-1s signaling and limiting cellular damage in response to stress. The proposed study is the first to target protein ufmylation in a model of cardiac failure and will employ unique tools including three new genetically-engineered mouse models to provide translational significance. Completion of this project will establish a novel role of post-translational modification (ufmylation) in the regulation of cardiac function and suggest new molecular targets for exploitation in the treatment of heart disease.